Ras inhibitor CAPRI enables neutrophil-like cells to chemotax through a higher-concentration range of gradients.

Neutrophils sense and migrate through an enormous range of chemoattractant gradients through adaptation. Here, we reveal that in human neutrophils, calcium-promoted Ras inactivator (CAPRI) locally controls the GPCR-stimulated Ras adaptation. Human neutrophils lacking CAPRI (caprikd ) exhibit chemoattractant-induced, nonadaptive Ras activation; significantly increased phosphorylation of AKT, GSK-3α/3ß, and cofilin; and excessive actin polymerization. caprikd cells display defective chemotaxis in response to high-concentration gradients but exhibit improved chemotaxis in low- or subsensitive-concentration gradients of various chemoattractants, as a result of their enhanced sensitivity. Taken together, our data reveal that CAPRI controls GPCR activation-mediated Ras adaptation and lowers the sensitivity of human neutrophils so that they are able to chemotax through a higher-concentration range of chemoattractant gradients.

Combined presentation and immunogenicity analysis reveals a recurrent RAS.Q61K neoantigen in melanoma.

Neoantigens are now recognized drivers of the antitumor immune response. Recurrent neoantigens, shared among groups of patients, have thus become increasingly coveted therapeutic targets. Here, we report on the data-driven identification of a robustly presented, immunogenic neoantigen that is derived from the combination of HLA-A*01:01 and RAS.Q61K. Analysis of large patient cohorts indicated that this combination applies to 3% of patients with melanoma. Using HLA peptidomics, we were able to demonstrate robust endogenous presentation of the neoantigen in 10 tumor samples. We detected specific reactivity to the mutated peptide within tumor-infiltrating lymphocytes (TILs) from 2 unrelated patients, thus confirming its natural immunogenicity. We further investigated the neoantigen-specific clones and their T cell receptors (TCRs) via a combination of TCR sequencing, TCR overexpression, functional assays, and single-cell transcriptomics. Our analysis revealed a diverse repertoire of neoantigen-specific clones with both intra- and interpatient TCR similarities. Moreover, 1 dominant clone proved to cross-react with the highly prevalent RAS.Q61R variant. Transcriptome analysis revealed a high association of TCR clones with specific T cell phenotypes in response to cognate melanoma, with neoantigen-specific cells showing an activated and dysfunctional phenotype. Identification of recurrent neoantigens and their reactive TCRs can promote "off-the-shelf" precision immunotherapies, alleviating limitations of personalized treatments.

Validation of Isoform- and Mutation-Specific RAS Antibodies.

Validation of antibody specificity is essential for the accurate evaluation of protein expression. For antibodies that recognize the gene products of the RAS family of oncogenes (HRAS, KRAS, and NRAS), an important challenge is the determination of selectivity for the four nearly identical HRAS, KRAS4A, KRAS4B, and NRAS proteins. With increasing appreciation for the distinct roles of the different RAS proteins in normal and neoplastic cells, there is a need for well-validated antibodies to evaluate the function and expression of the different RAS isoforms. Here we describe our experimental approaches to characterize RAS antibodies for their isoform- and mutant-specificity for use in immunoblot analyses.

Probing RAS Function with Monobodies.

RAS is frequently mutated in human cancers with nearly 20% of all cancers harboring mutations in one of three RAS isoforms (KRAS, HRAS, or NRAS). Furthermore, RAS proteins are critical oncogenic drivers of tumorigenesis. As such, RAS has been a prime focus for development of targeted cancer therapeutics. Although RAS is viewed by many as undruggable, the recent development of allele-specific covalent inhibitors to KRAS(G12C) has provided significant hope for the eventual pharmacological inhibition of RAS (Ostrem et al., Nature 503(7477):548-551, 2013; Patricelli et al., Cancer Discov 6(3):316-329, 2016; Janes et al., Cell 172(3):578-589.e17, 2018; Canon et al., Nature 575(7781):217-223, 2019; Hallin et al., Cancer Discov 10(1):54-71, 2020). Indeed, these (G12C)-specific inhibitors have elicited promising responses in early phase clinical trials (Canon et al., Nature 575(7781):217-223, 2019; Hallin et al., Cancer Discov 10(1):54-71, 2020). Despite this success in pharmacologically targeting KRAS(G12C), the remaining RAS mutants lack readily tractable chemistries for development of covalent inhibitors. Thus, alternative approaches are needed to develop broadly efficacious RAS inhibitors. We have utilized Monobody (Mb) technology to identify vulnerabilities in RAS that can potentially be exploited for development of novel RAS inhibitors. Here, we describe the methods used to isolate RAS-specific Mbs and to define their inhibitory activity.

Bispecific antibodies targeting mutant RAS neoantigens.

Mutations in the RAS oncogenes occur in multiple cancers, and ways to target these mutations has been the subject of intense research for decades. Most of these efforts are focused on conventional small-molecule drugs rather than antibody-based therapies because the RAS proteins are intracellular. Peptides derived from recurrent RAS mutations, G12V and Q61H/L/R, are presented on cancer cells in the context of two common human leukocyte antigen (HLA) alleles, HLA-A3 and HLA-A1, respectively. Using phage display, we isolated single-chain variable fragments (scFvs) specific for each of these mutant peptide-HLA complexes. The scFvs did not recognize the peptides derived from the wild-type form of RAS proteins or other related peptides. We then sought to develop an immunotherapeutic agent that was capable of killing cells presenting very low levels of these RAS-derived peptide-HLA complexes. Among many variations of bispecific antibodies tested, one particular format, the single-chain diabody (scDb), exhibited superior reactivity to cells expressing low levels of neoantigens. We converted the scFvs to this scDb format and demonstrated that they were capable of inducing T cell activation and killing of target cancer cells expressing endogenous levels of the mutant RAS proteins and cognate HLA alleles. CRISPR-mediated alterations of the HLA and RAS genes provided strong genetic evidence for the specificity of the scDbs. Thus, this approach could be applied to other common oncogenic mutations that are difficult to target by conventional means, allowing for more specific anticancer therapeutics.

Pan RAS-binding compounds selected from a chemical library by inhibiting interaction between RAS and a reduced affinity intracellular antibody.

Intracellular antibodies are valuable tools for target validation studies for clinical situations such as cancer. Recently we have shown that antibodies can be used for drug discovery in screening for chemical compounds surrogates by showing that compounds could be developed to the so-called undruggable RAS protein family. This method, called Antibody-derived compound (Abd) technology, employed intracellular antibodies binding to RAS in a competitive surface plasmon resonance chemical library screen. Success with this method requires a high affinity interaction between the antibody and the target. We now show that reduction in the affinity (dematuration) of the anti-active RAS antibody facilitates the screening of a chemical library using an in vitro AlphaScreen method. This identified active RAS specific-binding Abd compounds that inhibit the RAS-antibody interaction. One compound is shown to be a pan-RAS binder to KRAS, HRAS and NRAS-GTP proteins with a Kd of average 37 mM, offering the possibility of a new chemical series that interacts with RAS in the switch region where the intracellular antibody binds. This simple approach shows the druggability of RAS and is generally applicable to antibody-derived chemical library screening by affording flexibility through simple antibody affinity variation. This approach can be applied to find Abd compounds as surrogates of antibody-combining sites for novel drug development in a range of human diseases.

PI3Kγ Regulatory Protein p84 Determines Mast Cell Sensitivity to Ras Inhibition-Moving Towards Cell Specific PI3K Targeting?

Mast cells are the major effector cells in immunoglobulin E (IgE)-mediated allergy. The high affinity IgE receptor FcεRI, as well as G protein-coupled receptors (GPCRs) on the mast cell surface signals to phosphoinositide 3-kinase γ (PI3Kγ) to initiate degranulation, cytokine release, and chemotaxis. PI3Kγ is therefore considered as a target for treatment of allergic disorders. However, leukocyte PI3Kγ is key to many functions in innate and adaptive immunity, and attenuation of host defense mechanisms is an expected adverse effect that complicates treatment of chronic illnesses. PI3Kγ operates as a p110γ/p84 or p110γ/p101 complex, where p110γ/p84 requires Ras activation. Here we investigated if modulation of Ras-isoprenylation could target PI3Kγ activity to attenuate PI3Kγ-dependent mast cell responses without impairment of macrophage functions. In murine bone marrow-derived mast cells, GPCR stimulation triggers activation of N-Ras and H-Ras isoforms, which is followed by the phosphorylation of protein kinase B (PKB/Akt) relayed through PI3Kγ. Although K-Ras is normally not activated in Ras wild-type cells, it is able to compensate for genetically deleted N- and H-Ras isoforms. Inhibition of Ras isoprenylation with farnesyltransferase inhibitor FTI-277 leads to a significant reduction of mast cell degranulation, cytokine production, and migration. Complementation experiments expressing PI3Kγ adaptor proteins p84 or p101 demonstrated a differential sensitivity towards Ras-inhibition depending on PI3Kγ complex composition. Mast cell responses are exclusively p84-dependent and were effectively controlled by FTI-277. Similar results were obtained when GTP-Ras was inactivated by overexpression of the GAP-domain of Neurofibromin-1 (NF-1). Unlike mast cells, macrophages express p84 and p101 but are p101-dominated and thus remain functional under treatment with FTI-277. Our work demonstrates that p101 and p84 have distinct physiological roles, and that Ras dependence of PI3Kγ signaling differs between cell types. FTI-277 reduces GPCR-activated PI3Kγ  responses in p84-expressing but not p101-containing bone marrow derived cells. However, prenylation inhibitors have pleiotropic effects beyond Ras and non-tolerable side-effects that disfavor further clinical validation. Statins are, however, clinically well-established drugs that have previously been proposed to block mast cell degranulation by interference with protein prenylation. We show here that Simvastatin inhibits mast cell degranulation, but that this does not occur via Ras-PI3Kγ pathway alterations.

Therapeutic targeting of RAS: New hope for drugging the "undruggable".

RAS is the most frequently mutated oncogene in cancer and a critical driver of oncogenesis. Therapeutic targeting of RAS has been a goal of cancer research for more than 30 years due to its essential role in tumor formation and maintenance. Yet the quest to inhibit this challenging foe has been elusive. Although once considered "undruggable", the struggle to directly inhibit RAS has seen recent success with the development of pharmacological agents that specifically target the KRAS(G12C) mutant protein, which include the first direct RAS inhibitor to gain entry to clinical trials. However, the limited applicability of these inhibitors to G12C-mutant tumors demands further efforts to identify more broadly efficacious RAS inhibitors. Understanding allosteric influences on RAS may open new avenues to inhibit RAS. Here, we provide a brief overview of RAS biology and biochemistry, discuss the allosteric regulation of RAS, and summarize the various approaches to develop RAS inhibitors.

Comprehensive Analysis of ERK1/2 Substrates for Potential Combination Immunotherapies.

Recent clinical and therapeutic success with RAF and MEK1/2 inhibitors has revolutionized the existing treatment schemes for previously incurable cancers like melanomas. However, the overall therapeutic efficacies are still largely compromised by the dose-limiting side effects and emerging drug resistance mechanisms. Accumulating evidence has revealed the intricate nature of the RAS-RAF-MEK1/2-ERK1/2 pathway, such as activation mechanisms, kinase-substrate relationships, crosstalk with parallel signaling pathways, feedback regulations, and intimate interplay with immune responses. Limited strategies are currently available to exploit the benefits of combining RAF-MEK1/2-ERK1/2 pathway inhibitors with other targeted therapies or immunotherapies. Here, we compiled the kinase-substrate relationships and analyzed the intricate signaling networks of the renowned pathway, providing an integrated and simplified visualization, to reveal the potentials of RAS-RAF-MEK1/2-ERK1/2-based combination therapies.

The T helper type 17/regulatory T cell imbalance was associated with Ras-GTPase overexpression in patients with pulmonary hypertension associated with chronic obstructive pulmonary disease.

Pulmonary hypertension (PH) is a common but dangerous complication in chronic obstructive pulmonary disease (COPD). We hypothesized that dysregulation in the T helper type 17 (Th17) compartment could contribute to the development of COPD-associated PH (COPD-PH). To investigate this hypothesis, patients with COPD-PH and age- and sex-matched healthy controls were recruited, and their circulating CD4+ T cells were activated using anti-CD3/CD28 antibodies. The frequency of interleukin-17 (IL-17) -secreting cells was significantly higher in COPD-PH patients than in healthy controls. The secretion of IL-17 was significantly higher from COPD-PH CD4+ T cells than from control CD4+ T cells, whereas the secretion of interferon-γ and IL-4 was not significantly different. The expression of transforming growth factor-ß, on the other hand, was significantly higher in healthy controls than in COPD-PH patients. Activated CD4+ T cells from COPD-PH patients also presented significantly lower forkhead box P3 (FOXP3) and higher retinoic acid receptor-related orphan C2 (RORC2) expression than CD4+ T cells from healthy controls. In both controls and patients, a negative correlation between RORC2 and FOXP3 was found, ex vivo and after CD3/CD28 activation. The serum IL-6 level was slightly higher in COPD-PH patients than in controls, but the IL-6 transcription by monocytes was comparable in COPD-PH patients and controls. Interestingly, CD4+ T cells from COPD-PH patients presented significantly higher levels of Kirsten rat sarcoma viral oncogene homolog and neuroblastoma RAS viral oncogene homolog than CD4+ T cells from healthy controls. Inhibiting Ras-GTPases using farnesylthiosalicylic acid significantly reduced the ratio of RORC2/FOXP3 expression in CD4+ T cells. Overall, we demonstrated that an imbalance of Th17/regulatory T cells was a hallmark of COPD-PH.

Candida albicans Ras1 Inactivation Increases Resistance to Phagosomal Killing by Human Neutrophils.

Host phagocytic cells are crucial players in initial defense against Candida albicans infection. C. albicans utilizes MAP kinases and Ras1 stress response signaling pathways to protect itself from killing by immune cells. In this study, we tested the importance of these pathways in C. albicans phagocytosis by neutrophils and subsequent phagosomal survival. Phagocytosis was influenced by C. albicans morphology, so hyphal length of >10 µm reduced the phagocytic index (PI) 2- to 3-fold in human neutrophils. Primary human neutrophils killed 81% of phagocytosed C. albicans, while primary mouse neutrophils killed 63% of yeasts. We found that both the C. albicans Cek1 and Hog1 pathways were required for survival of phagocytosed yeast, whereas deletion of C. albicansRAS1 resulted in an 84% increase in survival within neutrophils compared to that of the wild type (WT). The absence of Ras1 did not alter reactive oxygen species (ROS) production by C. albicans; however, phagocytosed C. albicans Δ/Δras1 cells reduced ROS release by neutrophils by 86%. Moreover, C. albicans Δ/Δras1 cells had increased resistance to hydrogen peroxide as a result of high levels of catalase activity. This phenotype was specific to Ras1, since these effects were not observed in the absence of its partner Cyr1 or with its downstream target Efg1. In addition, C. albicans Δ/Δras1 cells had a significantly increased resistance to nonoxidative killing by human neutrophil peptide 1 (HNP-1) that was reversed by restoring cellular cAMP levels. These data show that C. albicans Ras1 inactivation leads to fungal resistance to both oxidative and nonoxidative mechanisms of neutrophil phagosomal killing.

Murine eosinophil development and allergic lung eosinophilia are largely dependent on the signaling adaptor GRB2.

Eosinophils are innate effector cells associated with allergic inflammation. Their development and survival is largely dependent on IL-5 and the common beta chain (ßc ) of the IL-5 receptor that serves as docking site for several proteins that mediate down-stream signaling cascades including JAK/STAT, PI3 kinase, NFκB, and RAS-MAP kinase pathways. The relative contribution of these signaling pathways for eosinophil development and homeostasis in vivo are poorly understood. Here, we investigated the role of GRB2, an adaptor protein that binds to ßc and other proteins and elicits the RAS-MAP kinase pathway. By using GRB2 inhibitors and inducible deletion of the Grb2 gene in mouse eosinophils we demonstrate that GRB2 plays a critical role for development of eosinophils from bone marrow precursors. Furthermore, Aspergillus fumigatus-induced allergic lung eosinophilia was significantly reduced in mice with induced genetic deletion of Grb2. Our results indicate that GRB2 is important for eosinophil development in steady-state conditions and during allergic inflammation. Based on these findings pharmacologic GRB2 inhibitors may have the potential to dampen tissue eosinophilia in various eosinophil-associated diseases.

Formaldehyde-mediated peptide coupling for the titration of epitope-specific antibody in an ELISA format.

Synthetic peptides are commonly used as an immobilised substrate in ELISA to measure antibody responses in serum. However, in addition to poor adsorption on the plastic surfaces of plates, titration of antibody response to a specific epitope of interest is challenging because the majority of antibodies in serum may be directed to other regions of the antigen. Using Ras peptides containing either the G12V or G12D mutation as a model antigen, we have tested formaldehyde as a crosslinker to immobilise Ras peptides on the plastic surface of microtitre plates in the presence of bovine serum albumin (BSA) as a carrier. Using this method, specific antibody against the G12V mutation was titrated from a simulated rabbit serum. Non-cognate Ras peptides were included in the ELISA reactions to suppress binding against non-mutated regions of the peptide. Our results showed that formaldehyde enhanced in-well peptide immobilisation of peptide approximately 3-fold and this enhancement required addition of BSA as a carrier. Wild type Ras peptide was not ideal as a non-cognate competitor as it tended to inhibit specific antibody binding against G12D or G12V. In the presence of the non-cognate peptide containing G12D, concentrations of the anti-G12V antibody in a simulated serum were determined with approximately 95% accuracy. Our method will be useful to determine a specific antibody response against a certain epitope in various peptide-based immunotherapy trials.

Evaluation of the selectivity and sensitivity of isoform- and mutation-specific RAS antibodies.

There is intense interest in developing therapeutic strategies for RAS proteins, the most frequently mutated oncoprotein family in cancer. Development of effective anti-RAS therapies will be aided by the greater appreciation of RAS isoform-specific differences in signaling events that support neoplastic cell growth. However, critical issues that require resolution to facilitate the success of these efforts remain. In particular, the use of well-validated anti-RAS antibodies is essential for accurate interpretation of experimental data. We evaluated 22 commercially available anti-RAS antibodies with a set of distinct reagents and cell lines for their specificity and selectivity in recognizing the intended RAS isoforms and mutants. Reliability varied substantially. For example, we found that some pan- or isoform-selective anti-RAS antibodies did not adequately recognize their intended target or showed greater selectivity for another; some were valid for detecting G12D and G12V mutant RAS proteins in Western blotting, but none were valid for immunofluorescence or immunohistochemical analyses; and some antibodies recognized nonspecific bands in lysates from "Rasless" cells expressing the oncoprotein BRAFV600E Using our validated antibodies, we identified RAS isoform-specific siRNAs and shRNAs. Our results may help to ensure the accurate interpretation of future RAS studies.

Structural and functional characterization of a DARPin which inhibits Ras nucleotide exchange.

Ras mutations are the oncogenic drivers of many human cancers and yet there are still no approved Ras-targeted cancer therapies. Inhibition of Ras nucleotide exchange is a promising new approach but better understanding of this mechanism of action is needed. Here we describe an antibody mimetic, DARPin K27, which inhibits nucleotide exchange of Ras. K27 binds preferentially to the inactive Ras GDP form with a Kd of 4 nM and structural studies support its selectivity for inactive Ras. Intracellular expression of K27 significantly reduces the amount of active Ras, inhibits downstream signalling, in particular the levels of phosphorylated ERK, and slows the growth in soft agar of HCT116 cells. K27 is a potent, non-covalent inhibitor of nucleotide exchange, showing consistent effects across different isoforms of Ras, including wild-type and oncogenic mutant forms.

R-Ras deficiency does not affect papain-induced IgE production in mice.

INTRODUCTION: R-Ras GTPase has recently been implicated in the regulation of immune functions, particularly in dendritic cell (DC) maturation, immune synapse formation, and subsequent T cell responses. METHODS: Here, we investigated the role of R-Ras in allergen-induced immune response (type 2 immune response) in Rras deficient (R-Ras KO) and wild type (WT) mice. RESULTS: Initially, we found that the number of conventional DC's in the lymph nodes (LNs) was reduced in R-Ras KO mice. The expression of co-stimulatory CD80 and CD86 molecules on these cells was also reduced on DC's from the R-Ras KO mice. However, there was no difference in papain-induced immune response between the R-Ras WT and KO as measured by serum IgE levels after the immunization. Interestingly, neither the DC number nor co-stimulatory molecule expression was different between WT and R-Ras KO animals after the immunization. CONCLUSIONS: Taken together, despite having reduced number of conventional DC's in the R-Ras KO mice and low expression of CD80 on DC's, the R-Ras KO mice are capable of mounting papain-induced IgE responses comparable to that of the WT mice. To our knowledge, this is the first report addressing potential differences in in vivo allergen responses regulated by the R-Ras GTPase.

Dysregulated Glycoprotein B-Mediated Cell-Cell Fusion Disrupts Varicella-Zoster Virus and Host Gene Transcription during Infection.

The highly conserved herpesvirus glycoprotein complex gB/gH-gL mediates membrane fusion during virion entry and cell-cell fusion. Varicella-zoster virus (VZV) characteristically forms multinucleated cells, or syncytia, during the infection of human tissues, but little is known about this process. The cytoplasmic domain of VZV gB (gBcyt) has been implicated in cell-cell fusion regulation because a gB[Y881F] substitution causes hyperfusion. gBcyt regulation is necessary for VZV pathogenesis, as the hyperfusogenic mutant gB[Y881F] is severely attenuated in human skin xenografts. In this study, gBcyt-regulated fusion was investigated by comparing melanoma cells infected with wild-type-like VZV or hyperfusogenic mutants. The gB[Y881F] mutant exhibited dramatically accelerated syncytium formation in melanoma cells caused by fusion of infected cells with many uninfected cells, increased cytoskeleton reorganization, and rapid displacement of nuclei to dense central structures compared to pOka using live-cell confocal microscopy. VZV and human transcriptomes were concurrently investigated using whole transcriptome sequencing (RNA-seq) to identify viral and cellular responses induced when gBcyt regulation was disrupted by the gB[Y881F] substitution. The expression of four vital VZV genes, ORF61 and the genes for glycoproteins gC, gE, and gI, was significantly reduced at 36 h postinfection for the hyperfusogenic mutants. Importantly, hierarchical clustering demonstrated an association of differential gene expression with dysregulated gBcyt-mediated fusion. A subset of Ras GTPase genes linked to membrane remodeling were upregulated in cells infected with the hyperfusogenic mutants. These data implicate gBcyt in the regulation of gB fusion function that, if unmodulated, triggers cellular processes leading to hyperfusion that attenuates VZV infection. IMPORTANCE: The highly infectious, human-restricted pathogen varicella-zoster virus (VZV) causes chickenpox and shingles. Postherpetic neuralgia (PHN) is a common complication of shingles that manifests as prolonged excruciating pain, which has proven difficult to treat. The formation of fused multinucleated cells in ganglia might be associated with this condition. An effective vaccine against VZV is available but not recommended for immunocompromised individuals, highlighting the need for new therapies. This study investigated the viral and cellular responses to hyperfusion, a condition where the usual constraints of cell membranes are overcome and cells form multinucleated cells. This process hinders VZV and is regulated by a viral glycoprotein, gB. A combination of live-cell imaging and next-generation genomics revealed an alteration in viral and cellular responses during hyperfusion that was caused by the loss of gB regulation. These studies reveal mechanisms central to VZV pathogenesis, potentially leading to improved therapies.

Diacylglycerol kinases in cancer.

Diacylglycerol kinases (DGK) are a family of enzymes that catalyze the transformation of diacylglycerol into phosphatidic acid. In T lymphocytes, DGKα and ζ limit the activation of the PLCγ/Ras/ERK axis, providing a critical checkpoint to inhibit T cell responses. Upregulation of these isoforms limits Ras activation, leading to hypo-responsive, anergic states similar to those caused by tumors. Recent studies have identified DGKα upregulation in tumor lymphocyte infiltrates, and cells from DGKα and ζ deficient mice show enhanced antitumor activity, suggesting that limitation of DAG based signals by DGK is used by tumors to evade immune attack. DGKα expression is low or even absent in other healthy cells like melanocytes, hepatocytes or neurons. Expression of this isoform, nevertheless is upregulated in melanoma, hepatocarcinoma and glioblastoma where DGKα contributes to the acquisition of tumor metastatic traits. A model thus emerges where tumor milieu fosters DGKα expression in tumors as well as in tumor infiltrating lymphocytes with opposite consequences. Here we review the mechanisms and targets that facilitate tumor "addiction" to DGKα, and discuss its relevance in the more advanced forms of cancer for tumor immune evasion. A better knowledge of this function offers a new perspective in the search of novel approaches to prevent inhibition of immune attack in cancer. Part of the failure in clinical progress may be attributed to the complexity of the tumor/T lymphocyte interaction. As they develop, tumors use a number of mechanisms to drive endogenous, tumor reactive T cells to a general state of hyporesponsiveness or anergy. A better knowledge of the molecular mechanisms that tumors use to trigger T cell anergic states will greatly help in the advance of immunotherapy research.

Adjuvant-dependent innate and adaptive immune signatures of risk of SIVmac251 acquisition.

A recombinant vaccine containing Aventis Pasteur's canarypox vector (ALVAC)-HIV and gp120 alum decreased the risk of HIV acquisition in the RV144 vaccine trial. The substitution of alum with the more immunogenic MF59 adjuvant is under consideration for the next efficacy human trial. We found here that an ALVAC-simian immunodeficiency virus (SIV) and gp120 alum (ALVAC-SIV + gp120) equivalent vaccine, but not an ALVAC-SIV + gp120 MF59 vaccine, was efficacious in delaying the onset of SIVmac251 in rhesus macaques, despite the higher immunogenicity of the latter adjuvant. Vaccine efficacy was associated with alum-induced, but not with MF59-induced, envelope (Env)-dependent mucosal innate lymphoid cells (ILCs) that produce interleukin (IL)-17, as well as with mucosal IgG to the gp120 variable region 2 (V2) and the expression of 12 genes, ten of which are part of the RAS pathway. The association between RAS activation and vaccine efficacy was also observed in an independent efficacious SIV-vaccine approach. Whether RAS activation, mucosal ILCs and antibodies to V2 are also important hallmarks of HIV-vaccine efficacy in humans will require further studies.

The antitumor efficacy of a novel adenovirus-mediated anti-p21Ras single chain fragment variable antibody on human cancers in vitro and in vivo.

Activated ras genes are found in a large number of human tumors, and therefore are one of important targets for cancer therapy. This study investigated the antitumor effects of a novel single chain fragment variable antibody (scFv) against ras protein, p21Ras. The anti-p21Ras scFv gene was constructed by phage display library from hybridoma KGHR1, and then subcloned into replication-defective adenovirus vector to obtain recombinant adenovirus KGHV100. Human tumor cell lines with high expression of p21Ras SW480, MDA-MB­231, OVCAR-3, BEL-7402, as well as tumor cell line with low expression of p21Ras, SKOV3, were employed to investigate antitumor effects in vitro and in vivo. Fluorescence microscopy demonstrated that KGHV100 was able to express intracellularly anti-p21Ras scFv antibody in cultured tumor cells and in transplantation tumor cells. MTT, Transwell, colony formation, and flow cytometry analysis showed that KGHV100 led to significant growth arrest in tumor cells with high p21Ras expression, and induced G0/G1 cell cycle arrest in the studied tumor cell lines. In vivo, KGHV100 significantly inhibited tumor growth following intratumoral injection, and the survival rates of the mice were higher than the control group. These results indicate that the adenovirus-mediated intracellular expression of the novel anti-p21Ras scFv exerted strong antitumoral effects, and may be a potential method for therapy of cancers with p21Ras overexpression.